Prodrugs of heteroaryl compounds

ABSTRACT

The present invention provides hydrophobic prodrugs of bases, nucleosides, and nucleotides as well as methods of using the prodrugs as antiviral and anti-cancer chemotherapeutic agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.12/419,959, filed Apr. 7, 2009, now U.S. Pat. No. 7,982,034, which is acontinuation of U.S. application Ser. No. 11/749,008, filed May 15,2007, now abandoned, which is a continuation of U.S. application Ser.No. 10/816,161, filed Mar. 31, 2004, now U.S. Pat. No. 7,244,732, whichclaims benefit of priority of U.S. Application No. 60/480,037, filedJun. 20, 2003; the disclosures of each application are hereinincorporated by reference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

Some of mankind's greatest medical threats are caused by viruses,including AIDS, hepatitis, rhinovirus infections of the respiratorytract, flu, measles, polio and others. There are a number of chronicpersistent diseases caused by RNA or DNA viruses, that replicate througha RNA intermediate, which are difficult to treat, such as hepatitis Band C, and HIV. A number of common human diseases are caused by RNAviruses that are replicated by a viral encoded RNA replicase. Includedin this group are influenza (Zurcher, et al., J. Gen. Virol. 77:1745(1996)), dengue fever (Becker, Virus-Genes 9:33 (1994)), and rhinovirusinfections (Horsnell, et al., J. Gen. Virol., 76:2549 (1995)). Animalsalso suffer from a wide variety of RNA viral diseases, including felineleukemia and immunodeficiency, Visna maedi of sheep, bovine viraldiarrhea, bovine mucosal disease, and bovine leukemia. Although somevaccines are available for DNA viruses, diseases such as hepatitis B arestill prevalent. Hepatitis B is caused by a DNA virus that replicatesits genome through a RNA intermediate (Summers and Mason, Cell 29:4003(1982)). While an effective vaccine exists as a preventive, treatmentfor chronic persistent Hepatitis B Viral (HBV) infection only cures aminority of patients.

Chain terminating nucleoside analogs have been used extensively for thetreatment of infections by DNA viruses and retroviruses. These analogshave been designed to be incorporated into DNA by DNA polymerases orreverse transcriptases. Once incorporated, they cannot be furtherextended and thus terminate DNA synthesis. Unfortunately, there isimmediate selective pressure for the development of resistance againstsuch chain terminating analogs that results in development of mutationsin the viral polymerase that prevent incorporation of the nucleosideanalog.

An alternative strategy is to utilize mutagenic deoxyribonucleosides(MDRN) or mutagenic ribonucleosides (MRN) that are preferentiallyincorporated into a viral genome. MDRN are incorporated into DNA byviral reverse transcriptase or by a DNA polymerase enzyme. MRN areincorporated into viral RNAs by viral RNA replicases. As a result, themutations in the viral genome affect all viral proteins by creatinginactive versions of them. These mutations are perpetuated andaccumulated with each viral replication cycle. Eventually, through thesheer number of mutations, a gene which is necessary for the function,replication, or transfection of the virus will be inactivated which willcease the viral life cycle. Because MDRN and MRN are not specificallytargeting one particular viral protein, there is less likelihood for thedevelopment of resistance.

5-aza-2′-deoxycytidine (5-aza-dC) is an antineoplastic agent that hasbeen tested in patients with leukemia and is thought to actpredominantly by demethylating DNA. Methylation is thought to silencetumor growth suppressor and differentiation genes. Interestingly,5-aza-dC affects other targets. For example, 5-aza-dC was shown toinhibit HIV replication in vitro, although the mechanism of action wasnot determined (see e.g., Bouchard et al, Antimicrob. Agents Chemother.34:206-209 (2000)). Deamination of 5-aza-dC to 5-aza-2′-deoxyuridine(5-aza-dU) has been shown to result in loss of antineoplastic activity(see e.g., Momparler, et al., Leukemia. 11:1-6 (1997)).

While 5-azacytidine (5-aza-C) or 5-aza-dC and variants thereof showpromise as MDRNs and MRNs, these compounds are also unstable and rapidlydegrade upon reconstitution. For example, at pH 7.0, a 10% degradationin 5-aza-dC occurs at temperatures of 25° C. and 50° C. after 5 and 0.5hours, respectively (see e.g., Van Groeningen et al., Cancer Res.46:4831-4836 (1986)). Thus, therapeutic use of these compounds islimited for treatment of both viral diseases and cancer. The presentinvention solves this and other problems.

BRIEF SUMMARY OF THE INVENTION

The present invention provides hydrophobic prodrugs of bases,nucleosides, and nucleotides as well as methods of using the prodrugs asantiviral and anti-cancer chemotherapeutic agents. Thus, in a firstaspect, the compounds of the invention have a structure according toFormula I:

in which a is either 0 or 1. The dashed line represents a double bondbetween C* and N when a is 0. The symbol R² is a member selected from(═O) and NR⁷R⁸. R⁴ is a member selected from H, halogen, OR³, NR⁷R⁸,halogen, nitrile, and substituted and unsubstituted (C₁-C₅)alkyl. Thesymbol R⁶ is a member selected from H, halogen, OR³, NR³R³, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted 3- to 7-membered cycloalkyl, substituted orunsubstituted 5- to 7-membered heterocycloalkyl, substituted orunsubstituted acyl, substituted or unsubstituted aryl, and substitutedor unsubstituted heteroaryl. R⁷, R⁸, R⁵ and R¹ are members independentlyselected from H, OR³, NR³R³, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstituted3- to 7-membered cycloalkyl, substituted or unsubstituted 5- to7-membered heterocycloalkyl, substituted or unsubstituted acyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl. The symbol R³ is independently selected from H, substitutedor unsubstituted alkyl and substituted or unsubstituted acyl. The groupsR⁷ and R⁸, R⁸ and R⁵, and R⁵ and R⁶, together with the atoms to whichthey are joined, optionally form a substituted or unsubstituted 5- to7-membered ring. In the compounds of the invention, at least one memberselected from R³, R⁵, R⁷, and R⁸, alone or together with the atom towhich it is covalently bonded, is selected from carbamate and urealinkers.

In a second aspect, the compounds of Formula I are used to treat a viraldisease through administering a therapeutically effective amount of thecompound to a patient in need of such treatment.

In a third aspect of the present invention, the compounds of Formula Iare used to treat cancer through administering a therapeuticallyeffective amount of the compound to a patient in need of such treatment.

In a fourth aspect, the present invention provides a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and acompound of Formula I.

These and other aspects, objects and advantages of the present inventionwill be apparent from the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 displays structures of representative compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The invention is directed to compounds which inhibit viral replicationand the growth of cancerous cells. These compounds are hydrophobicprodrugs of bases, nucleosides, and nucleotides. The compounds areeasily synthesized and comprise a parent compound, a hydrophobic group,and a linker which covalently attaches the parent compound to thehydrophobic group. Addition of the hydrophobic group and linkersignificantly improves the compound's pharmacokinetic properties overthe unmodified parent compound.

The compounds of the invention are useful for inhibiting viralreplication in cell culture as well as in antiviral therapy for animalsand humans. In one embodiment, the compounds and methods of theinvention are advantageous when used to target RNA viruses (viruses witha RNA genome), and retroviruses or other viruses otherwise replicated bya RNA intermediate. In another embodiment, the compounds and methods ofthe invention are advantageous for targeting DNA viruses (viruses with aDNA genome) such as hepatitis B virus, herpesviruses, and papillomaviruses. In one embodiment, the compounds are incorporated into bothviral encoded and cellular encoded viral genomic polynucleotidesequences, thereby causing miscoding in progeny copies of the genomicvirus, e.g., by tautomerism, which allows base mispairing (See, e.g.,Moriyama et al., Nucleic Acids Symp. Ser. 42:131-132 (1999); Robinson etal., Biochemistry 37:10897-10905 (1998); Anensen et al., Mutat. Res.476:99-107 (2001); Lutz et al., Bioorg. Med. Chem. Lett. 8:499-504(1998); and Klungland et al., Toxicology Lett. 119:71-78 (2001)).

The compounds of the invention are useful for inhibiting the growth ofcancer cells in cell culture as well as in treating cancer in animalsand humans. In an exemplary embodiment, the cancer is a hematopoieticcancer, such as leukemia or lymphoma. In some embodiments, the prodrugsare efficiently incorporated into the bloodstream of the animal or humanand, subsequently, into the polynucleotide sequence (either DNA or RNA)of a cancerous cell. The compounds of the invention have alteredbase-pairing properties which allow incorporation of mutations into thegenome of the cancer cell, dramatically reducing the ability of thecancer cell to efficiently replicate its genome. In another embodiment,mutations are incorporated into transcription products, such as mRNAmolecules or tRNA molecules, dramatically reducing the ability of thecancer cell to encode active proteins. As a result of these mutations,the cancer cells will either die, have diminished growth rates, or beunable to proliferate or metastasize.

II. Definitions

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—; —NHS(O)₂— is also intended to represent. —S(O)₂HN—, etc.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e., C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The team “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.”

The term “alkylene”, by itself or as part of another substituent, meansa divalent radical derived from an alkane, as exemplified, but notlimited, by —CH₂CH₂CH₂CH₂—, and further includes those groups describedbelow as “heteroalkylene.” Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

“Acyl” refers to a moiety that is a residue of a carboxylic acid fromwhich an oxygen atom is removed, i.e., —C(O)R, in which R is substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl or substituted or unsubstituted heteroaryl.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, S and Si, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N, S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,typically aromatic, hydrocarbon substituent, which can be a single ringor multiple rings (up to three rings), which are fused together orlinked covalently. The term “heteroaryl” refers to aryl groups (orrings) that contain from zero to four heteroatoms selected from N, O,and S, wherein the nitrogen and sulfur atoms are optionally oxidized,and the nitrogen atom(s) are optionally quaternized. A heteroaryl groupcan be attached to the remainder of the molecule through a heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R∝″, —NR″C(O)₂R′, —NR—C(NR′R″R″″)═NR″″,—NR—C(NR′R″)═NR″″,

—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂ in a numberranging from zero to (2 m′+1), where m′ is the total number of carbonatoms in such radical. R′, R″, R′″ and R″″ each preferably independentlyrefer to hydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., arylsubstituted with 1-3 halogens, alkoxy or thioalkoxy groups, or arylalkylgroups. When a compound of the invention includes more than one R group,for example, each of the R groups is independently selected as are eachR′, R″, R′″ and R″″ groups when more than one of these groups ispresent. When R′ and R″ are attached to the same nitrogen atom, they canbe combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″,—SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R∝″, —NR″C(O)₂R′,—NR—C(NR′R″)═NR″″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system. R′, R″, R′″ and R″″ each preferablyindependently refer to hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, e.g., aryl substituted with 1-3 halogens, alkoxy or thioalkoxygroups, or arylalkyl groups. When a compound of the invention includesmore than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r), —B—, wherein A and B are independently —CRR′—,—O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r isan integer of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S) and silicon (Si).

“Moiety” refers to the radical of a molecule that is attached to anotherstructure.

The symbol “R” is a general abbreviation that represents a substituentgroup that is selected from substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, and substituted orunsubstituted heterocyclyl groups.

“Reactive functional group,” as used herein refers to groups including,but not limited to, olefins, acetylenes, alcohols, phenols, ethers,oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides,cyanates, isocyanates, thiocyanates, isothiocyanates, amines,hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro, nitriles,mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids,sulfinic acids, acetals, ketals, anhydrides, sulfates, sulfenic acids,isonitriles, amidines, imides, imidates, nitrones, hydroxylamines,oximes, hydroxamic acids, thiohydroxamic acids, allenes, ortho esters,sulfites, enamines, ynamines, ureas, pseudoureas, semicarbazides,carbodiimides, carbamates, imines, azides, azo compounds, azoxycompounds, and nitroso compounds. Reactive functional groups alosinclude those used to prepare bioconjugates, e.g., N-hydroxysuccinimideesters, maleimides and the like. Methods to prepare each of thesefunctional groups are well known in the art and their application to ormodification for a particular purpose is within the ability of one ofskill in the art (see, for example, Sandler and Karo, eds. ORGANICFUNCTIONAL GROUP PREPARATIONS, Academic Press, San Diego, 1989).

“Protecting group,” as used herein refers to a portion of a substratethat is substantially stable under a particular reaction condition, butwhich is cleaved from the substrate under a different reactioncondition. A protecting group can also be selected such that itparticipates in the direct oxidation of the aromatic ring component ofthe compounds of the invention. For examples of useful protectinggroups, see, for example, Greene et al., PROTECTIVE GROUPS IN ORGANICSYNTHESIS, John Wiley & Sons, New York, 1991.

The symbol

, whether utilized as a bond or displayed perpendicular to a bondindicates the point at which the displayed moiety is attached to theremainder of the molecule, solid support, etc.

The term “compounds of the invention” encompass hydrophobic prodrugs, aswell as the unmodified parent compounds of the hydrophobic prodrugs.

The term “prodrug” comprises derivatives of active drugs which have beenmodified by the addition of a chemical group. This chemical groupusually reduces or eliminates the drug's biological activity while, atthe same time, conferring some other property to the drug. Once thechemical group has been cleaved from the prodrug, by hydrolysis,reduction, oxidation, light, heat, cavitation, pressure, and/or enzymesin the surrounding environment, the active drug is generated. Prodrugsmay be designed as reversible drug derivatives and utilized as modifiersto enhance drug transport to site-specific tissues. Prodrugs aredescribed in the art, for example, in R. L. Juliano (ed.), BIOLOGICALAPPROACHES TO THE CONTROLLED DELIVERY OF DRUGS, Annals of the New YorkAcademy of Sciences, Vol 507 (1998); Hans Bundgaard (ed.), DESIGN OFPRODRUGS, Elsevier Science, (1986); and Kenneth Sloan (ed.), PRODRUGS:TOPICAL AND OCULAR DRUG DELIVERY, Drugs and the Pharmaceutical Sciences,Vol 53 (1992).

The term “pharmaceutically acceptable salts” includes salts of theactive compounds which are prepared with relatively nontoxic acids orbases, depending on the particular substituents found on the compoundsdescribed herein. When compounds of the present invention containrelatively acidic functionalities, base addition salts can be obtainedby contacting the neutral form of such compounds with a sufficientamount of the desired base, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When compounds of the present invention containrelatively basic functionalities, acid addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired acid, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable acid addition salts includethose derived from inorganic acids like hydrochloric, hydrobromic,nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al., “Pharmaceutical Salts”, Journal ofPharmaceutical Science 66:1-19 (1997)). Certain specific compounds ofthe present invention contain both basic and acidic functionalities thatallow the compounds to be converted into either base or acid additionsalts.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents, but otherwise the salts are equivalentto the parent form of the compound for the purposes of the presentinvention.

In addition to salt forms, the present invention provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention can exist in tautomericforms. In general, all tautomeric forms are equivalent and areencompassed within the scope of the present invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are encompassed within thescope of the present invention.

The compounds of the invention may be prepared as a single isomer (e.g.,enantiomer, cis-trans, positional, diastereomer) or as a mixture ofisomers. In an exemplary embodiment, the compounds are prepared assubstantially a single isomer. Methods of preparing substantiallyisomerically pure compounds are known in the art. For example,enantiomerically enriched mixtures and pure enantiomeric compounds canbe prepared by using synthetic intermediates that are enantiomericallypure in combination with reactions that either leave the stereochemistryat a chiral center unchanged or result in its complete inversion.Alternatively, the final product or intermediates along the syntheticroute can be resolved into a single stereoisomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose andappropriate method for a particular situation. See, generally, Furnisset al. (eds.), VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY5^(TH) ED., Longman Scientific and Technical Ltd., Essex, 1991, pp.809-816; and Heller, Acc. Chem. Res. 23:128 (1990).

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

The term “viral disease” refers to a condition caused by a virus. Aviral disease is caused by a DNA virus, a RNA virus, or a retrovirus.

The term “vitamin K” refers to 2-methyl-1,4-naphthoquinone andderivatives thereof that have coagulation activity. The natural formsare substituted in position 3 of the quinone with an alkyl side chain.Examples of vitamin K include Vitamin K₁ (phylloquinones), and vitaminK₂ (menaquinones).

As used herein, the term “base” encompasses aryl and heteroarylstructures which are capable of covalent attachment to a sugar moiety.Examples include naturally-occurring bases such as adenine, guanine,cytosine, thymine and uracil. “Bases” also include non-natural bases,such as nitroindole, 5-aza-cytosine, and dihydro-5-aza-cytosine.

As used herein, the term “nucleoside” includes both the naturallyoccurring nucleosides (adenosine, guanosine, cytidine, thymidine, anduridine) and modifications thereof. Modifications include, but are notlimited to, those providing chemical groups that incorporate additionalcharge, polarizability, hydrogen bonding, and electrostatic interactionto the nucleosides. Such modifications include, but are not limited to,peptide nucleic acids (PNAs), 2′-position sugar modifications,5-position pyrimidine modifications, 8-position purine modifications,modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo or 5-iodo-uracil; backbone modifications,methylations, isobases, such as isocytidine and isoguanidine and thelike. “Nucleosides” can also include non-natural bases, such as, forexample, nitroindole, 5-aza-cytidine, 5-aza-2′-deoxycytidine, anddihydro-5-aza-2′-deoxycytidine. Modifications can also includederivitization with a quencher, a fluorophore or another moiety.“Nucleotides” are phosphate esters of nucleosides. Many of the chemicalreactions which are utilized for nucleosides can also be utilized fornucleotides.

As used herein, “nucleic acid” encompasses bases, nucleosides, andnucleotides, and modifications thereof. Examples of modifications arelisted in the definition of “nucleosides” above.

A “polynucleotide sequence” is a deoxyribonucleotide or ribonucleotidepolymer in either single- or double-stranded form. Unless otherwiselimited, “polynucleotide sequence” encompasses analogs of naturalnucleotides.

A “genomic polynucleotide sequence” is a nucleotide polymer which ishomologous to naturally occurring polynucleotide sequences (RNA or DNA)which are packaged by a viral particle. Typically, the packagedpolynucleotide sequence encodes some or all of the components necessaryfor viral replication. The genomic polynucleotide sequence optionallyincludes nucleotide analogs. Polynucleotide sequences are homologouswhen they are derived from a polynucleotide sequence with a commonsequence (an “ancestral” polynucleotide sequence) by natural orartificial modification of the ancestral polynucleotide sequence.Retroviral genomic polynucleotide sequences optionally encode a RNAwhich is competent to be packaged by a retroviral particle. Suchpolynucleotide sequences can be constructed by recombinantly combining apackaging site with a polynucleotide sequence of choice.

A “virally infected cell” is a cell transduced with a viralpolynucleotide sequence. The polynucleotide sequence is optionallyincorporated into the cellular genome, or is optionally episomal.

The “mutation rate” of a virus or polynucleotide sequence refers to thenumber of changes which occur upon copying the polynucleotide sequence,e.g., by a polymerase. Typically, this is measured over time, i.e., thenumber of alterations which occur during rounds of copying orgenerations of virus.

A “polymerase” refers to an enzyme that produces a polynucleotidesequence (DNA or RNA) which is complementary to a pre-existingpolynucleotide template (DNA or RNA). For example, a RNA polymerase maybe a RNA polymerase (viral or cellular) or a replicase. The polymerasemay be either naturally occurring, or artificially (e.g., recombinantly)produced.

A “cell culture” is a population of cells residing outside of an animal.These cells are optionally primary cells (isolated from a cell bank,animal, or blood bank), secondary cells (cultured from one of the abovesources), or long-lived, artificially maintained, in vitro cultures.

A “progressive loss of viability” refers to a measurable reduction inthe replicative or infective ability of a population of viruses overtime or in response to treatment with a prodrug of the invention.

A “viral particle” is genetic material substantially encoded by a RNAvirus or a virus with a RNA intermediate, such as BVDV, HCV, or HIV. Thepresence of non-viral or cellular components in the particle is a commonresult of the replication process of a virus, which typically includesbudding from a cellular membrane.

An “HIV particle” is a retroviral particle substantially encoded by HIV.The presence of non-HIV viral or cellular components in the particle isa common result of the replication process of HIV which typicallyincludes budding from a cellular membrane. In certain applications,retroviral particles are deliberately “pseudotyped” by co-expressingviral proteins from more than one virus (often HIV and vesicularstomatitis virus (VSV)) to expand the host range of the resultingretroviral particle. The presence or absence of non-HIV components in anHIV particle does not change the essential nature of the particle, i.e.,the particle is still produced as a primary product of HIV replication.

As used herein, “cancer” includes solid tumors and hematologicalmalignancies. The former includes cancers such as breast, colon, andovarian cancers. The latter include hematopoietic malignancies includingleukemias, lymphomas and myelomas. This invention provides new effectivemethods and compositions for treatment and/or prevention of varioustypes of cancer.

The term “patient” refers to any warm-blooded animal, such as a mouse,rat, dog, or human.

A “pharmaceutically acceptable” component is one that is suitable foruse in a patient without undue adverse side effects (such as toxicity,irritation, and allergic response) commensurate with a reasonablebenefit/risk ratio.

A “safe and effective amount”, or a “therapeutically effective amount”,refers to the quantity of a component that is sufficient to yield adesired therapeutic response without undue adverse side effects (such astoxicity, irritation, or allergic response). In some embodiments, thedesired therapeutic response is enhancing mutagenesis of a virus,diminishing the ability of a virus to produce active proteins,inhibiting replication of a virus, eliminating or diminishing theability of a virus to produce infectious particles, or killing the virusor a virally infected cell. In other embodiments, the therapeuticresponse is halting or delaying the growth of a cancer, or causing acancer to shrink, or not to metastasize. The specific safe and effectiveamount or therapeutically effective amount will vary with such factorsas the particular condition being treated, the physical condition of thepatient, the type of patient being treated, the duration of thetreatment, the nature of concurrent therapy (if any), the specificformulations employed, and the structure of the compounds or itsderivatives.

III. The Compounds

The compounds of the invention are hydrophobic prodrugs of bases,nucleosides, and nucleotides. These prodrugs comprise a parent compound,a hydrophobic group, and a linker which covalently attaches the parentcompound to the hydrophobic group. Addition of the hydrophobic group andlinker significantly improves the pharmacokinetic properties of theprodrug relative to the unmodified parent compounds.

The compounds of the invention are easily synthesized from commerciallyavailable starting materials and reagents. Synthetic schemesillustrating the preparation of the compounds are located in part B ofthis section. Detailed synthetic protocols and characterization data aresupplied in Example 1.

A. Prodrug Components

i) Parent Compound

The prodrugs of the invention comprise a parent compound. In someembodiments, the parent compound is a base, nucleoside, or nucleotide.In other embodiments, the parent compound is a cytosine analog. In someembodiments, the cytosine analog is a derivative of 5-aza-cytosine,5-aza-dC, and DHAdC. In other embodiments, the cytosine analog has astructure according to Formula I:

in which a is either 0 or 1. The dashed line represents a double bondbetween C* and N when a is 0. The symbol R² is a member selected from(═O) and NR⁷R⁸. R⁴ is a member selected from H, halogen, OR³, NR⁷R⁸,halogen, nitrile, and substituted and unsubstituted (C₁-C₅)alkyl. Thesymbol R⁶ is a member selected from H, halogen, OR³, NR³R³, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted 3- to 7-membered cycloalkyl, substituted orunsubstituted 5- to 7-membered heterocycloalkyl, substituted orunsubstituted acyl, substituted or unsubstituted aryl, and substitutedor unsubstituted heteroaryl. R⁷, R⁸, R⁵ and R¹ are members independentlyselected from H, OR³, NR³R³, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstituted3- to 7-membered cycloalkyl, substituted or unsubstituted 5- to7-membered heterocycloalkyl, substituted or unsubstituted acyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl. The symbol R³ is independently selected from H, substitutedor unsubstituted alkyl and substituted or unsubstituted acyl. The groupsR⁷ and R⁸, R⁸ and R⁵, and R⁵ and R⁶, together with the atoms to whichthey are joined, optionally form a substituted or unsubstituted 5- to7-membered ring. In the compounds of the invention, at least one memberselected from R³, R⁵, R⁷, and R⁸, alone or together with the atom towhich it is covalently bonded, is selected from carbamate and urealinkers.

In an exemplary embodiment, R¹ comprises a hydroxyl moiety. In anotherexemplary embodiment, R¹ comprises a saccharyl moiety. In yet anotherexemplary embodiment, R¹ is a structure according to Formula II:

in which the dashed line represents a double bond between C^(a) andC^(b). R⁹, R¹⁰ and R¹¹ are members independently selected from H, —OH,—OR¹², —NH₂, —NO₂, —SO₂NH₂, N₃, halogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted 3- to 7-membered cycloalkyl, substituted or unsubstituted5- to 7-membered heterocycloalkyl, substituted or unsubstituted acyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl. The symbol R¹² is selected from an amino acid and a peptidecomprising between 2 and 5 amino acids. The symbols R⁹ and R¹⁰ and R¹⁰and R¹¹, together with the atoms to which they are joined, optionallyform a substituted or unsubstituted 5- to 7-membered ring.

In another exemplary embodiment, the symbols R⁹, R¹⁰ and R¹¹ are membersindependently selected from H, OH, (R¹³)₃SiO—, and a structure accordingto Formula III:

in which each R¹³ is independently selected from substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted 3- to 7-membered cycloalkyl, substituted orunsubstituted 5- to 7-membered heterocycloalkyl, substituted orunsubstituted acyl, substituted or unsubstituted aryl, and substitutedor unsubstituted heteroaryl. More than one R¹³, together with the atomsto which they are joined, optionally form a substituted or unsubstituted5- to 7-membered ring. The symbols R¹⁶, R¹⁷, and R¹⁸ are independentlyselected from substituted and unsubstituted alkyl. In another exemplaryembodiment, the symbols R¹⁶, R¹⁷, and R¹⁸ are ethyl.

ii) Hydrophobic Group

The prodrugs of the invention also comprise a hydrophobic group. Thisgroup should improve the prodrug's absorption in the body anddistribution to organs by increasing its lipophilicity. C₆-C₁₀ alkylgroups, lipophilic vitamins, and cholesterol are three exemplaryhydrophobic groups.

In some embodiments, the hydrophobic group is R¹⁴ from Formula IV. R¹⁴is selected from substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted acyl, substituted or unsubstituted aryl, and substitutedor unsubstituted heteroaryl, an amino acid, and a peptide comprisingbetween 2 and 5 amino acids. In another exemplary embodiment, R¹⁴ isselected from substituted or unsubstituted (C₄-C₁₂)alkyl, benzyl,2-nitro-furanyl, retinol, α-tocopherol, calciferol, vitamin K,cholesterol,

In yet another exemplary embodiment, R¹⁴ is substituted or unsubstituted(C₆-C₁₀)alkyl. In still another exemplary embodiment, R¹⁴ isunsubstituted (C₆-C₁₀)alkyl.

In other embodiments, the chemical group is R¹⁵ from Formula V. R¹⁵ isselected from substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted acyl, substituted or unsubstituted aryl, and substitutedor unsubstituted heteroaryl, an amino acid, and a peptide comprisingbetween 2 and 5 amino acids. In another exemplary embodiment, R¹⁵ isselected from substituted or unsubstituted (C₄-C₁₂)alkyl, benzyl,2-nitro-furanyl, retinol, α-tocopherol, calciferol, vitamin K,cholesterol,

In yet another exemplary embodiment, R¹⁵ is substituted or unsubstituted(C₆-C₁₀)alkyl. In still another exemplary embodiment, R¹⁵ isunsubstituted (C₆-C₁₀)alkyl.

iii) Linker

The prodrug also contains a linker which covalently attaches thehydrophobic group to the parent compound. This linker should be stableunder certain conditions, and yet labile in others. For example, orallyavailable compounds preferably include a linker that is stable underacidic conditions and yet labile under the enzymatic conditions thatexist in the liver. Carbamate groups and urea groups are two exemplarylinkers.

In an exemplary embodiment, a structure according to Formula I containsa carbamate or urea linker. In yet another exemplary embodiment, astructure according to Formula I has at least one member selected fromR³, R⁵, R⁷, and R⁸, which, either alone or together with the nitrogenatom to which it is covalently bonded, is either a carbamate or a urea.

In an exemplary embodiment, R³, R⁵, R⁷, and R⁸ are independentlyselected from H and a structure according to Formula IV or V:

If R⁸ is a structure according to either Formula IV or Formula V, thenR⁷ is H.

iv) Pharmacokinetic Properties

The covalent attachment of a hydrophobic group and linker to a parentcompound significantly improves its pharmacokinetics relative to itsunmodified form. Pharmacokinetics are improved either through anincrease or a decrease in certain pharmacokinetic properties.

In some embodiments, prodrug pharmacokinetics are improved, relative tothose of the parent compound, through an increase in a pharmacokineticproperty selected from half-life (t_(1/2)), oral bioavailability,lipophilicity, stability in acidic pH or uptake from thegastrointestinal tract. In some embodiments, the pharmacokineticproperty is increased by an amount between 10% and 600%. In someembodiments, the pharmacokinetic property is increased by an amountbetween 10% and 100%. In some embodiments, the pharmacokinetic propertyis increased by an amount between 400% and 500%. In some embodiments,the half-life is increased by an amount between 200% and 500%. In someembodiments, the oral bioavailability is increased by an amount between200% and 400%.

In other embodiments, prodrug pharmacokinetics are improved, relative toits unmodified form, through a decrease in a pharmacokinetic propertysuch as a reduction in the first-pass effect. In some embodiments, thefirst-pass effect is reduced by an amount between 10% and 100%. In otherembodiments, the first-pass effect is reduced by an amount between 400%and 500%.

The improved pharmacokinetic property is observed in a patient selectedfrom a mouse, rat, dog, or human.

Pharmacokinetic data for the compounds of the invention have beenacquired in rat and dog models. The laboratory protocol for the ratstudy is provided in Example 2, while the laboratory protocol for thedog study is provided in Example 4. In both studies, DHAdC was theparent compound, the hydrophobic group was a C₇-C₉ unsubstituted alkylchain, and the linker was a carbamate moiety. Oral administrations ofthe DHAdC prodrugs were tested against IV and oral administrations ofthe parent compound. The results of the rat study are provided inExample 3, while the results of the dog study are provided in Example 5.

In the rat study, both prodrugs had half lives which were between300-400% longer than DHAdC. In addition, the prodrugs reached thecirculatory system (as evidenced by Oral Bioavailability) in amountsbetween 200-300% greater than DHAdC. Because the prodrug versions ofDHAdC are more effective at reaching the target cells than DHAdC alone,these compounds are more effective viral and cancer inhibitors.

The laboratory protocol for the lipophilicity study is provided inExample 6, while the lipophilicity data for the compounds of theinvention are described in Example 7.

B. Prodrug Preparation

The following exemplary schemes 1-7 illustrate methods of preparing thecompounds of the invention. These methods are not limited to producingthe compounds listed, but can be used to prepare other compounds aswell. The compounds of the invention can also be produced by methods notexplicitly illustrated in the schemes. The compounds can be preparedusing readily available starting materials or known intermediates.

The carbonyl of compound 1 can be protected according to the method ofScheme 1.

In this scheme, a trimethylsilyl protecting group is added to thecarbonyl of commercially available 5-azacytosine (CAS #: 931-86-2, SigmaChemical Company).

Compound 2 can be attached to a protected saccharide according to themethod of Scheme 2.

In Scheme 2, compound 2 is reacted with compound 3 in dichloromethane toproduce compound 4. Compound 4 represents a mixture of approximately 70%β-anomer 4a and 30% α-anomer 4b.

The double bond at the 5-position in compound 4 can be reduced withsodium borohydride according to the method of Scheme 3.

In Scheme 3, compound 4 is reacted with sodium borohydride in aceticacid to produce compound 5. Compound 5 represents a mixture ofapproximately 70% β-anomer 5a and 30% α-anomer 5b.

The pure β-anomer 5a is separated from the α-anomer 5b throughrecrystallization in methanol. The protecting groups are removed fromcompound 5a through the method according to Scheme 4.

Adding sodium methoxide to compound 5a in methanol produces thedeprotected compound 6.

A mixture of exo and endo N-acylated products can be produced accordingto the method of Scheme 5.

In Scheme 5, the hydroxyl groups of compound 6 are first protected usingTMS chloride to form compound 7. Subsequently, the treatment of 7 withone equivalent of the appropriate chloroformate produces a mixture ofendo-N-acylated product 8a and exo-N-acylated product 8b. The mainendo-N-acylated product 8a is separated from the exo-N-acylated isomer8b by flash chromatography on silica gel.

The pure exo-N-acylated isomer can be obtained by the method of Scheme6.

In Scheme 6, compounds 8a and 8b are first treated with an excess of achloroformate to create the bis-N-acylated compound 9. Subsequentdeprotection of the endo-N-acylated moiety from compound 9 withtriethylamine in methanol produces the exo-N-acylated isomer 10 in ahigh overall yield.

The endo-N-acylated isomer 14 can be obtained frombenzyloxycarbonyl-protected DHAdC 11 by the method of Scheme 7.

In Scheme 7, compound 7 is first treated with an excess ofbenzyloxychloroformate to create the bis-N-acylated compound 11.Subsequent deprotection of the endo-N-acylated moiety from compound 11with triethylamine in methanol produces the exo-N-acylated isomer 12.Treatment with an amount of chloroformate will create compound 13.Catalytic hydrogenation of compound 13 produces the endo-N-acylatedisomer 14.

IV. The Viruses

The compounds of the invention possess activity against viruses. Some ofthese viruses are able to integrate their viral genome into the genomeof a cell. Examples of viruses which have this ability include, but arenot limited to, retroviruses. In an exemplary embodiment, the virus isHIV and its variants, such as HIV-1, HIV-2, HTLV-1, HTLV-II, and SIV. Inanother embodiment, the virus is a DNA virus such as hepatitis B virus,herpesviruses (e.g., Herpes Simplex Virus, CytoMegaloVirus (CMV),Epstein-Barr Virus, (EBV)), smallpox virus, or human papilloma virus(e.g., HPV). Alternatively, the viral genome can be episomal. Theseinclude many human and animal pathogens: flaviviruses, such as denguefever, West Nile, and yellow fever; pestiviruses, such as bovine viraldiarrhea (BVD), and hepaciviruses, such as hepatitis C; filoviruses suchas ebola; parainfluenza viruses, including respiratory syncytial;rubulaviruses, such as mumps; morbillivirus, such as measles;picornaviruses, including the echoviruses; the coxsackieviruses; thepolioviruses; the togaviruses, including encephalitis; coronaviruses,including Severe Acute Respiratory Syndrome (SARS); rubella;bunyaviruses; reoviruses, including rotaviruses; rhabdoviruses;arenaviruses, such as lymphocytic choriomeningitis, as well as other RNAviruses of man and animal.

Retroviruses that can be targeted include HTLV viruses such as HTLV-1and HTLV-2, adult T-cell leukemia (ATL), HIV-1 and HIV-2 and SIV. Insome embodiments, the HIV virus is resistant to non-nucleoside reversetranscriptase inhibitors. In certain embodiments, the virus is hepatitisA or hepatitis B. See, Knipe et al. FIELDS VIROLOGY, 4th ed. Lippincott,Williams, and Wilkins (2001). Further information regarding viraldiseases and their replication can be found in White and Fenner, MEDICALVIROLOGY, 4th ed. Academic Press (1994) and in Zuckerman, Banatvala andPattison (ed.), PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY, John Wileyand Sons (1994).

V. Methods of Treating Viral Diseases

The compounds, methods, and pharmaceutical compositions of the presentinvention are useful in the treatment of viral diseases. In one aspect,the invention provides a method of treating a viral disease comprisingadministering to a subject in need of such treatment a therapeuticallyeffective amount of a compound according to Formula I. In an exemplaryembodiment, the viral disease is caused by a virus that is a memberselected from a RNA virus and a DNA virus. In another exemplaryembodiment, the virus is selected from a retrovirus and a ribovirus. Inyet another exemplary embodiment, the retrovirus is selected from HIVand Hepatitis B. In still another exemplary embodiment, the ribovirus isHepatitis C.

In one embodiment for the treatment of viral diseases, the compounds ofthe invention are efficiently delivered into the bloodstream of apatient, such as a mouse, rat, dog or human, and subsequentlyincorporated into the genome of the virus of interest. The compounds ofthe invention either have phosphodiester linkages or acquirephosphodiester linkages, enabling them to be incorporated into the viralgenome by a polymerase. In some embodiments, the compounds of theinvention have altered base-pairing properties which allow theincorporation of mutations into the viral genome, thereby increasing thetotal number of mutations. Increases in the total number of mutationsresult in reduced viral population growth rates, as well as decreasedviability of progeny virus.

Methods of Treating HIV

The compounds of the invention are useful for treating HIV infectionsand other retroviral infections. The compounds of the present inventionare particularly well-suited to treat HIV strains that are resistant tochain-terminating nucleosides. In one embodiment, compounds of theinvention are used for treating an HIV strain which is resistant to achain-terminating nucleoside.

HIV strains resistant to chain-terminating nucleosides are known andmutations in the reverse transcriptase (RT) enzyme responsible for theresistance have been analyzed. Two mechanisms of viral resistance towardchain-terminating nucleosides have been described. In the firstmechanism, the virus discriminates between a chain-terminatingnucleoside and a naturally occurring nucleoside, thus preventing thechain-terminating nucleoside's incorporation into the viral genome. Forexample, chain-terminating nucleoside-resistant viral strains contain aversion of HIV-RT which recognizes the absence of a 3′-OH group, afeature present in some chain-terminating nucleosides (see, e.g.,Sluis-Cremer et al., Cell. Mol. Life. Sci. 57:1408-1422 (2000)). In thesecond mechanism, the virus excises the chain-terminating nucleosideafter its incorporation into the viral genome via pyrophosphorolysis inthe presence of nucleotides (see, e.g., Isel et al., J. Biol. Chem.276:48725-48732 (2001)). In pyrophosphorolysis, also known as reversenucleotide polymerization, pyrophosphate acts as an acceptor moleculefor the removal of the chain-terminating nucleoside. Removal of thechain-terminating nucleoside frees RT to incorporate the naturalnucleotide substrate and maintain accurate viral replication. ATP hasalso been proposed as an acceptor molecule for the removal ofchain-terminating nucleosides and is referred to as primer unblocking(see, e.g., Naeger et al., Nucleosides Nucleotides Nucleic Acids20:635-639 (2001)).

The compounds of the invention can reduce viral resistance through thefirst mechanism mentioned above. Because the compounds of the inventioncomprise sugars with hydroxyls at the 3′ position, it is believed thatHIV-RT should be unable to differentiate between them and naturalnucleosides.

In general, the compounds of the invention will reduce viral resistancecompared to treatment with chain-terminating nucleosides. Currentlyapproved chain-terminating nucleosides target one aspect of the viralgrowth cycle, replication, and immediately attempt to stop it throughchain termination. Since the antiviral's effect is narrowly targeted andabrupt, there is great selective pressure for the development ofresistant viral strains. The compounds of the invention act by adifferent method. The compounds act through the gradual accumulation ofrandom mutations in the viral genome. This corresponds to the gradualinactivation of potentially any of the viral proteins. Since the effectof the compounds of the invention is broadly targeted and gradual, thereis less selective pressure for the emergence of resistant viral strains.

Cross resistance between chain-terminating nucleosides and the compoundsof the invention can be tested by determining the EC₅₀ for a prodrug ina wild-type HIV strain and in a HIV strain resistant to one or morechain-terminating nucleosides. If the EC₅₀ for the prodrug is higher inthe chain-terminating nucleoside resistant strain than in the wild-typestrain, then cross resistance has occurred. Experiments havedemonstrated that cross resistance is unlikely to develop betweenchain-terminating nucleosides and compounds of the invention.

VI. Cancer

The compounds of the invention possess activity against cancer. In someembodiments, the prodrugs possess activity against hematologicalmalignancies. Hematological malignancies, such as leukemias andlymphomas, are conditions characterized by abnormal growth andmaturation of hematopoietic cells.

Leukemias are generally neoplastic disorders of hematopoietic stemcells, and include adult and pediatric acute myeloid leukemias (AML),chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL),chronic lymphocytic leukemia (CLL), hairy cell leukemia and secondaryleukemia. Myeloid leukemias are characterized by infiltration of theblood, bone marrow, and other tissues by neoplastic cells of thehematopoietic system. CLL is characterized by the accumulation ofmature-appearing lymphocytes in the peripheral blood and theinfiltration of these mature-appearing lymphocytes into the bone marrow,spleen and lymph nodes.

Specific leukemias include acute nonlymphocytic leukemia, chroniclymphocytic leukemia, acute granulocytic leukemia, chronic granulocyticleukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemicleukemia, aleukocythemic leukemia, basophylic leukemia, blast cellleukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis,embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cellleukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocyticleukemia, stem cell leukemia, acute monocytic leukemia, leukopenicleukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocyticleukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cellleukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblasticleukemia, monocytic leukemia, myeloblastic leukemia, myelocyticleukemia, myeloid granulocytic leukemia, myelomonocytic leukemia,Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia,promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stemcell leukemia, subleukemic leukemia, and undifferentiated cell leukemia.

Lymphomas are generally neoplastic transformations of cells that resideprimarily in lymphoid tissue. Among lymphomas, there are two majordistinct groups: non-Hodgkin's lymphoma (NHL) and Hodgkin's disease.Lymphomas are tumors of the immune system and generally involve both T-and B-cells. Lymphomas are typically found in bone marrow, lymph nodes,the spleen and the circulatory system. Treatment protocols includeremoval of bone marrow from the patient, purging the bone marrow oftumor cells (often using antibodies directed against antigens present onthe tumor cell type), followed by storage of the bone marrow. After thepatient receives a toxic dose of radiation or chemotherapy, the purgedbone marrow is reinfused in order to repopulate the patient'shematopoietic system.

Other hematological malignancies include myelodysplastic syndromes(MDS), myeloproliferative syndromes (MPS) and myelomas, such as multiplemyeloma and solitary myeloma. Multiple myeloma (also called plasma cellmyeloma) affects the skeletal system and is characterized by multipletumorous masses of neoplastic plasma cells scattered throughout thesystem. It may also spread to lymph nodes and other sites such as theskin. Solitary myeloma involves solitary lesions that tend to occur inthe same locations as multiple myeloma.

The compounds of the invention are also directed against other cancers.Such cancers include those characterized by solid tumors. Examples ofother cancers of concern are skin cancers, including melanomas, basalcell carcinomas, and squamous cell carcinomas. Epithelial carcinomas ofthe head and neck are also encompassed by the present invention. Thesecancers typically arise from mucosal surfaces of the head and neck andinclude salivary gland tumors.

The present invention also encompasses cancers of the lung. Lung cancersinclude squamous or epidermoid carcinoma, small cell carcinoma,adenocarcinoma, and large cell carcinoma. Breast cancer is alsoincluded.

The present invention also encompasses gastrointestinal tract cancers.Gastrointestinal tract cancers include esophageal cancers, gastricadenocarcinoma, primary gastric lymphoma, colorectal cancer, small boweltumors and cancers of the anus. Pancreatic cancer and cancers thataffect the liver are also of concern, including hepatocellular cancer.The present invention also includes treatment of bladder cancer andrenal cell carcinoma.

The present invention also encompasses prostatic carcinoma andtesticular cancer.

Gynecologic malignancies are also encompassed by the present inventionand include ovarian cancer, carcinoma of the fallopian tube, uterinecancer, and cervical cancer.

Treatment of sarcomas of the bone and soft tissue are encompassed by thepresent invention. Bone sarcomas include osteosarcoma, chondrosarcoma,and Ewing's sarcoma.

The present invention also encompasses malignant tumors of the thyroid,including papillary, follicular, and anaplastic carcinomas.

VII. Methods of Treating Cancer

The compounds, methods, and pharmaceutical compositions of the inventionare useful in the treatment of cancer. In one aspect, the inventionprovides a method of treating cancer comprising administering to asubject in need of such treatment a therapeutically effective amount ofa compound according to Formula I. In an exemplary embodiment, thecancer is a leukemia, lymphoma, or other hematopoietic cancer.

In one embodiment for the treatment of cancer, the compounds of theinvention are efficiently delivered into the bloodstream of a patient,such as a mouse, rat, dog or human, and subsequently incorporated into apolynucleotide sequence (either DNA or RNA) of a cancerous cell. In someembodiments, the compounds of the invention have phosphodiester linkagesor can acquire phosphodiester linkages, allowing them to be incorporatedinto the genome of a cancer cell by a polymerase. In another embodiment,the compounds of the invention have altered base-pairing properties andare incorporated into the cancer cell genome. Incorporation subsequentlyincreases the number of mutations in the cancer cell. In anotherembodiment, mutations are incorporated into transcription products,e.g., mRNA molecules that encode proteins or tRNA molecules useful forprotein translation. The mutated transcription products possess alteredamino acid sequences which often result in inactive proteins. Regardlessof the method of introduction, an increase in the number of mutations inthe cancer cell causes reduced population growth rates, decreasedviability of progeny cells, diminished ability to proliferate ormetastasize, and cancer cell death.

Those of skill in the art are aware of methods to test the effectivenessof compounds in treating cancer. For example, cancer cells of interestcan be grown in culture and incubated in the presence of varyingconcentrations of the compounds of the present invention. Frequently,the uptake of viral dyes, such as MTT, is used to determine cellviability and cell proliferation. When inhibition of cell proliferationis seen, the IC₅₀ of the compound can be determined. Those of skill inthe art will also know to test the compounds of the present invention inanimal models. For example, the compounds of the invention are injectedinto nude mice with transformed cancer cells. The data gathered intissue culture models and animal models can be extrapolated by those ofskill in the art for use in human patients.

VIII. Assays for Detecting Compounds of the Invention

A. Assays for Mutagenic Nucleic Acids

Nucleic acids are incorporated into the genome of a virus or a cell withan efficiency of at least about 0.1%. In some cases, the incorporationis at least about 5%, and most preferably equal to that of a naturallyoccurring complementary polynucleotide sequence when compared in equalamounts in an in vitro assay. Thus, an error rate of about 1 in 1000bases or more would be sufficient to enhance mutagenesis of the virus.The ability of a nucleic acid to cause incorrect base pairing may bedetermined by testing and examining the frequency and nature ofmutations produced by the incorporation of a compound of the inventioninto DNA or RNA. These mutation rates can vary widely. It has beenreported, for example, that the mutation rates in lytic RNA viruses(such as influenza A) are about 300 times higher than in DNA viruses(Drake, Proc. Natl. Acad. Sci. USA 90:4171-4175 (1993)). Retroviruses,however, have mutation rates that are an order of magnitude lower, onaverage, than lytic RNA viruses.

Assays for the incorporation rates of altered nucleotides are analogousto those used for incorporation of deoxynucleoside triphosphates by DNApolymerases (Boosalis, et al., J. Biol. Chem. 262:14689-14698 (1987)).Those of skill in the art will recognize that such assays measure acompound's ability to inhibit a cellular polymerase or measure thereplicative capability of a virus that has been treated with an alterednucleotide. In selected situations direct determination of the frequencyof mutations that are introduced into the viral genome (Ji and Loeb,Virol., 199:323-330 (1994)) can be made.

For example, in the case of HIV, the viral RNA or the incorporated HIVDNA is isolated and then copied using reverse transcriptase PCR(RT-PCR).The region of the genome copied corresponds to a 600 nucleotide segmentin the reverse transcriptase gene. After 70 rounds of RT-PCR, the copiedDNA or RNA is treated with restriction enzymes and ligated into aplasmid. After transfection of the plasmid into E. coli, individualclones are obtained and the amplified segment within the plasmid issequenced. Mutations within this region are determined by computer aidedanalysis, comparing the individual sequences with control viralsequences obtained by parallel culturing of the same virus in theabsence of the RNA analog. For each nucleotide, determinations arecarried out after ten sequential rounds of viral passage or at the pointof extinction for viral detection. Analogous procedures would beeffective for other viruses of interest and would be readily apparent tothose of skill in the art.

A comparison of incorporation of compounds of the invention among thepolymerases of interest can be carried out using a modification of the“minus” sequencing gel assay for nucleotide incorporation. A5′-³²P-labeled primer is extended in a reaction containing three of thefour nucleoside triphosphates and a compound of the invention intriphosphate form. The template can be either RNA or DNA, asappropriate. Elongation of the primer past the template nucleotide thatis complementary to the nucleotide that is omitted from the reactionwill depend upon, and be proportional to, the incorporation of theanalog. The degree of analog incorporation is calculated as a functionof the percent of oligonucleotide that is extended on the sequencing gelfrom one position to the next. Incorporation is determined byautoradiography followed by either densitometry or cutting out each ofthe bands and counting radioactivity by liquid scintillationspectroscopy. Those of skill in the art will recognize that similarexperiments can be done to determine the incorporation of the compoundsof the invention into polynucleotide sequences in cancer cells.

When a compound of the invention is administered to virally infectedcells, either in vitro or in vivo, a population of cells is producedcomprising a highly variable population of replicated homologous viralpolynucleotide sequences. This population of highly variable cellsresults from administering mutagenic compounds of the invention tovirally infected cells and increasing the mutation rate of the viruspopulation. Thus, the highly variable population of viruses is anindicator that the mutation rate of the virus was increased by theadministration of the compounds of the invention. Measuring thevariability of the population provides an assessment of the viability ofthe viral population. In turn, the viability of the viral population isa prognostic indicator for the health of the cell population. Forexample, low viability for an HIV population in a human patientcorresponds to an improved outlook for the patient.

In some embodiments, the mutagenic compound of the invention will bewater soluble and have the ability to rapidly enter the target cells.Lipid soluble analogs are also encompassed by the present invention. Ifnecessary, the compounds of the invention are phosphorylated by cellularkinases and incorporated into RNA or DNA.

B. Assays of Viral Replication

Those of skill in the art recognize that viral replication orinfectivity correlates with the ability of a virus to cause disease.That is, a highly infectious virus is more likely to cause disease thana less infectious virus. In a preferred embodiment, a virus that hasincorporated mutations into its genome as a result of treatment with thecompounds of this invention will have diminished viral infectivitycompared to untreated virus. Those of skill in the art are aware ofmethods to assay the infectivity of a virus. (See, e.g., Condit,Principles of Virology, in FIELDS VIROLOGY, 4th Ed. 19-51 (Knipe et al.,eds., 2001)).

For example, a plaque forming assay can be used to measure theinfectivity of a virus. Briefly, a sample of virus is added to anappropriate medium and serial dilutions are plated onto confluentmonolayers of cells. The infected cells are overlaid with a semisolidmedium so that each plaque develops from a single viral infection. Afterincubation, the plates are stained with an appropriate dye so thatplaques can be visualized and counted.

Some viruses do not kill cells, but rather transform them. Thetransformation phenotype can be detected by, for example, formation offoci after loss of contact inhibition. The virus is serially diluted andplated onto monolayers of contact inhibited cells. Foci can be detectedwith an appropriate dye and counted to determine the infectivity of thevirus.

Another method to determine virus infectivity is the endpoint method.The method is appropriate for viruses that do not form plaques or foci,but that do have a detectable pathology or cytopathic effect (CPE) incultured cells, embryonated eggs, or animals. A number of phenotypes aremeasurable as CPEs, including rounding, shrinkage, increasedrefractility, fusion, syncytia formation, aggregation, loss of adherenceor lysis. Serial dilutions of virus are applied to an appropriate assaysystem and after incubation, CPE is assayed. Statistical methods areavailable to determine the precise dilution of virus required forinfection of 50% of the cells. (See, e.g., Spearman, Br. J. Psychol.2:227-242 (1908); and Reed and Muench, Am. J. Hyg. 27:493-497 (1938)).

Measurements of viral replication can also be performed indirectly dueto the difficulty in culturing viruses. For example, a replicon assay,which measures the inhibition of a self replicating genetic element, canbe used to determine the extent of a virus's replication. HIV viralreplication can be determined from measuring levels of p24 antigen. Oneexemplary means to determine antiviral activity is with CEM-SS cells andvirus (e.g., HIV-1_(RF)) (MOI=0.01) using the XTT(2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide) cytoprotection assay (see, e.g., Weislow, et al, J. Natl.Canc. Inst. 81:577-586 (1989); Rice PNAS 90:9721-9724 (1993); and RiceAntimicrob. Agents Chemother. 41:419-426 (1997)). Briefly, cells areinfected with HIV-1_(RF) (or other virus to be tested) in the presenceof various dilutions of the compounds of the invention. The cultures areincubated for seven days. During this time control cultures withoutprotective compounds (i.e., compounds with anti-viral activity)replicate virus, induce syncytia, and result in about 90% cell death.The cell death is measured by XTT dye reduction. XTT is a solubletetrazolium dye that measures mitochondrial energy output, similar toMTT. Positive controls, including dextran sulfate (an attachmentinhibitor), 3′-Azido-2′-3′-dideoxythymidine, or AZT (a reversetranscriptase inhibitor), are added to each assay. Individual assays aredone in duplicate using a sister plate method.

The ability of a drug to inhibit viral replication or infectivity isexpressed as the EC₅₀ of the drug, or the effective concentration thatprevents 50% of viral replication. Methods described above to determinethe infectivity of a virus are useful to determine the EC₅₀ of a drug.

The ability of a drug to kill cells is expressed as the IC₅₀, or theconcentration of drug that inhibit cellular proliferation. Methods todetermine the IC₅₀, of a drug are known to those of skill in the art andinclude determination of cell viability after incubation with a range ofconcentrations of the drug.

IX. Pharmaceutical Compositions of the Invention

The present invention provides pharmaceutical compositions which inhibitthe replication of viruses and the growth of cancer cells. Thesepharmaceutical compositions comprise a prodrug of a base, nucleoside, ornucleotide and a pharmaceutically acceptable carrier. In one embodimentof the invention, the pharmaceutical compositions comprise prodrugs ofcytosine and a pharmaceutically acceptable carrier. In anotherembodiment, the pharmaceutical compositions comprise a compoundaccording to Formula I and a pharmaceutically acceptable carrier.

A pharmaceutical composition of the invention, or pharmaceuticallyacceptable addition salt or hydrate thereof, can be delivered to apatient using a wide variety of routes or modes of administration.Suitable routes of administration include, but are not limited to, oral,transdermal, transmucosal (such as intranasal or intravaginal), andparenteral administration, including intramuscular, subcutaneous andintravenous injections.

In an exemplary embodiment, the present invention provides a method oftreating a viral disease or treating cancer by administering thecompound orally.

A. Oral Administration

For oral administration, the compounds can be formulated readily bycombining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained by combining the composition with a suitablesolid phase excipient, optionally grinding the resulting mixture, andprocessing the mixture of granules, after adding suitable auxiliaries,if desired, to obtain tablets or dragee cores. Suitable excipients are,for example, calcium carbonate, calcium phosphate, polymers such aspoly(ethylene oxide), fillers such as sugars, including lactose,sucrose, mannitol, or sorbitol; cellulose preparations such as, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, poly(ethyleneoxide), and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations, which can be used orally, include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

B. Parenteral Administration

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such ascross-linked polyvinyl pyrrolidone, agar, or alginic acid or a saltthereof such as sodium alginate. For injection, the agents of theinvention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiological saline buffer. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents, which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated can be used in theformulation. Such penetrants are generally known in the art, andinclude, e.g., for transmucosal administration, bile salts and fusidicacid derivatives. In addition, detergents can be used to facilitatepermeation. Transmucosal administration can be through nasal sprays, forexample, or using suppositories.

For topical administration, the agents are formulated into ointments,creams, salves, powders and gels. In one embodiment, the transdermaldelivery agent can be DMSO. In another embodiment, the transdermaldelivery agent can be a transdermal patch. The compounds may beformulated, for example, with suitable polymeric or hydrophobicmaterials (e.g., as an emulsion in an acceptable oil) or ion exchangeresins, or as sparingly soluble derivatives, for example, as a sparinglysoluble salt.

Examples of aqueous solutions that can be used in formulations fortransmucosal drug delivery include, e.g., water, saline, phosphatebuffered saline, Hank's solution, Ringer's solution, dextrose/saline,glucose solutions and the like. The formulations can containpharmaceutically acceptable auxiliary substances to enhance stability,deliverability or solubility, such as buffering agents, tonicityadjusting agents, wetting agents, detergents and the like. Additives canalso include additional active ingredients such as bactericidal agents,or stabilizers. For example, the solution can contain sodium acetate,sodium lactate, sodium chloride, potassium chloride, calcium chloride,sorbitan monolaurate or triethanolamine oleate. These compositions canbe sterilized by conventional, well-known sterilization techniques, orcan be sterile filtered. The resulting aqueous solutions can be packagedfor use as is, or lyophilized, the lyophilized preparation beingcombined with a sterile aqueous solution prior to administration.

The choice of therapeutic agents that can be co-administered with thecompounds of the invention will depend, in part, on the condition beingtreated. For example, when administered to a patient undergoing cancertreatment, the compounds may be administered in cocktails containingother bioactive agents, such as anti-cancer agents and/or supplementarypotentiating agents. The compounds may also be administered in cocktailscontaining agents that treat the side-effects of radiation therapy, suchas anti-emetics, radiation protectants, etc.

Other suitable bioactive agents include, for example, antineoplasticagents, such as platinum compounds (e.g., spiroplatin, cisplatin, andcarboplatin), methotrexate, adriamycin, taxol, mitomycin, ansamitocin,bleomycin, cytosine arabinoside, arabinosyl adenine, mercaptopolylysine,vincristine, busulfan, chlorambucil, melphalan (e.g., PAM, L-PAM orphenylalanine mustard), mercaptopurine, mitotane, procarbazinehydrochloride dactinomycin (actinomycin D), daunorubicin hydrochloride,doxorubicin hydrochloride, mitomycin, plicamycin (mithramycin),aminoglutethimide, estramustine phosphate sodium, flutamide, leuprolideacetate, megestrol acetate, tamoxifen citrate, testolactone, trilostane,amsacrine (m-AMSA), asparaginase (L-asparaginase) Erwina asparaginase,etoposide (VP-16), interferon α-2a, interferon α-2b, teniposide (VM-26),vinblastine sulfate (VLB), vincristine sulfate, bleomycin, bleomycinsulfate, methotrexate, adriamycin, and arabinosyl; blood products suchas parenteral iron, hemin, hematoporphyrins and their derivatives;biological response modifiers such as muramyldipeptide,muramyltripeptide, microbial cell wall components, lymphokines (e.g.,bacterial endotoxin such as lipopoly-saccharide, macrophage activationfactor), sub-units of bacteria (such as Mycobacteria andCorynebacteria), the synthetic dipeptideN-acetyl-muramyl-L-alanyl-D-isoglutamine; anti-fungal agents such asketoconazole, nystatin, griseofulvin, flucytosine (5-fc), miconazole,amphotericin B, ricin, and β-lactam antibiotics (e.g., sulfazecin);hormones and steroids such as growth hormone, melanocyte stimulatinghormone, estradiol, beclomethasone dipropionate, betamethasone,betamethasone acetate and betamethasone sodium phosphate, vetamethasonedisodium phosphate, vetamethasone sodium phosphate, cortisone acetate,dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate,flunsolide, hydrocortisone, hydrocortisone acetate, hydrocortisonecypionate, hydrocortisone sodium phosphate, hydrocortisone sodiumsuccinate, methylprednisolone, methylprednisolone acetate,methylprednisolone sodium succinate, paramethasone acetate,prednisolone, prednisolone acetate, prednisolone sodium phosphate,prednisolone tebutate, prednisone, triamcinolone, triamcinoloneacetonide, triamcinolone diacetate, triamcinolone hexacetonide andfludrocortisone acetate; vitamins such as cyanocobalamin neinoic acid,retinoids and derivatives such as retinol palmitate, and α-tocopherol;peptides, such as manganese super oxide dimutase; enzymes such asalkaline phosphatase; anti-allergic agents such as amelexanox;anti-coagulation agents such as phenprocoumon and heparin; circulatorydrugs such as propranolol; metabolic potentiators such as glutathione;antituberculars such as para-aminosalicylic acid, isoniazid, capreomycinsulfate cycloserine, ethambutol hydrochloride ethionamide, pyrazinamide,rifampin, and streptomycin sulfate; antivirals such as acyclovir,amantadine azidothymidine (AZT or Zidovudine), ribavirin, amantadine,vidarabine, and vidarabine monohydrate (adenine arabinoside, ara-A);antianginals such as diltiazem, nifedipine, verapamil, erythrityltetranitrate, isosorbide dinitrate, nitroglycerin (glyceryl trinitrate)and pentaerythritol tetranitrate; anticoagulants such as phenprocoumonand heparin; antibiotics such as dapsone, chloramphenicol, neomycin,cefaclor, cefadroxil, cephalexin, cephradine, erythromycin, clindamycin,lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin,dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin,nafcillin, oxacillin, penicillin G, penicillin V, ticarcillin rifampinand tetracycline; antiinflammatories such as diffinisal, ibuprofen,indomethacin, meclofenamate, mefenamic acid, naproxen, oxyphenbutazone,phenylbutazone, piroxicam, sulindac, tolmetin, aspirin and salicylates;antiprotozoans such as chloroquine, hydroxychloroquine, metronidazole,quinine and meglumine antimonate; antirheumatics such as penicillamine;narcotics such as paregoric; opiates such as codeine, heroin, methadone,morphine and opium; cardiac glycosides such as deslanoside, digitoxin,digoxin, digitalin and digitalis; neuromuscular blockers such asatracurium besylate, gallamine triethiodide, hexafluorenium bromide,metocurine iodide, pancuronium bromide, succinylcholine chloride(suxamethonium chloride), tubocurarine chloride and vecuronium bromide;sedatives (hypnotics) such as amobarbital, amobarbital sodium,aprobarbital, butabarbital sodium, chloral hydrate, ethchlorvynol,ethinamate, flurazepam hydrochloride, glutethimide, methotrimeprazinehydrochloride, methyprylon, midazolam hydrochloride, paraldehyde,pentobarbital, pentobarbital sodium, phenobarbital sodium, secobarbitalsodium, talbutal, temazepam and triazolam; local anesthetics such asbupivacaine hydrochloride, chloroprocaine hydrochloride, etidocainehydrochloride, lidocaine hydrochloride, mepivacaine hydrochloride,procaine hydrochloride and tetracaine hydrochloride; general anestheticssuch as droperidol, etomidate, fentanyl citrate with droperidol,ketamine hydrochloride, methohexital sodium and thiopental sodium; andradioactive particles or ions such as strontium, iodide rhenium andyttrium. In certain preferred embodiments, the bioactive agent is amonoclonal antibody, such as a monoclonal antibody capable of binding toa melanoma antigen.

Frequency of administration of the therapeutic compositions describedherein, as well as dosage, will vary from individual to individual, andmay be readily established using standard techniques. Preferably,between 1-100 doses may be administered over a 52-week period. Whentreating a viral disease, a suitable dose is an amount of a compoundthat, when administered as described above, is capable of killing orlimiting the infectivity of a virus. When treating cancer, a suitabledose is an amount of a compound that, when administered as describedabove, is capable of killing or slowing the growth of cancers or cancercells. Those of skill in the art are aware of the routineexperimentation that will produce an appropriate dosage range for apatient in need of treatment by oral administration or any other methodof administration of a drug, e.g., intravenous administration orparenteral administration, for example. Those of skill are also awarethat results provided by in vitro or in vivo experimental models can beused to extrapolate approximate dosages for a patient in need oftreatment.

In general, an appropriate dosage and treatment regimen provides thepharmaceutical composition in an amount sufficient to providetherapeutic and/or prophylactic benefit. Such a response can bemonitored by establishing an improved clinical outcome (e.g., longerviral disease-free survival or, for cancer patients, more frequentremissions or complete, partial, or longer disease-free survival) intreated patients as compared to non-treated patients.

All references and patent publications referred to herein are herebyincorporated by reference herein. As can be appreciated from thedisclosure provided above, the present invention has a wide variety ofapplications. Accordingly, the following examples are offered forillustration purposes and are not intended to be construed as alimitation on the invention in any way.

EXAMPLES General

In the examples below, unless otherwise stated, temperatures are givenin degrees Celsius (° C.); operations were carried out at room orambient temperature, “rt,” or “RT,” (typically a range of from about18-25° C.); evaporation of solvent was carried out using a rotaryevaporator under reduced pressure (typically, 4.5-30 mm Hg) with a bathtemperature of up to 60° C.; the course of reactions was typicallyfollowed by TLC and reaction times are provided for illustration only;melting points are uncorrected; products exhibited satisfactory ¹H-NMRand/or microanalytical data; yields are provided for illustration only;and the following conventional abbreviations are also used: mp (meltingpoint), L (liter(s)), mL (milliliters), mmol (millimoles), g (grams), mg(milligrams), min (minutes), and h (hours).

Unless otherwise specified, all solvents (HPLC grade) and reagents werepurchased from suppliers and used without further purification.Reactions were conducted under a blanket of argon unless otherwisestated. Analytical thin layer chromatography (TLC) was performed onplates from EM Science (Silica Gel 60F₂₅₄, 0.25 mm thickness) using a(12:1:1:1) ethyl acetate:acetone:water:methanol mixture. Compounds werevisualized under UV lamp (254 nM) or by developing with 3NH₂SO₄/0.5%cysteine solution followed by heating. Flash chromatography was doneusing silica gel from Whatman Inc. 60 Å silica gel (particle size230-400 mesh). ¹H NMR spectra were recorded on a Varian 300 machine at300 MHz. LC MS analysis was performed on a ThermoFinnigan LCQ Advantagemachine using a 0.01 M ammonium acetate:MeCN gradient and UV and ESI fordetection. Melting points were recorded on a Electrothermal 1101DMEL-TEMP apparatus and were uncorrected.

Example 1 Preparation ofN⁴-alkyloxycarbonyl-2′-deoxy-5,6-dihydro-5-azacytidines

1.1 General Procedure

β-2′-Deoxy-5,6-dihydro-5-azacytidine (40.2 g, 0.175 mol) was suspendedin 850 mL of pyridine and cooled with ice. The mixture was treated withchlorotrimethylsilane (72 mL, 0.570 mol) and kept on ice for 40 min.Corresponding alkylchloroformate (0.350 mol) was added to the mixture,and the mixture was kept on ice for 2 hrs. The mixture was treated withmethanol (200 mL) and evaporated under vacuum. The residue was takeninto ethyl acetate (1 L) and washed with water (0.5 L). The extract waswashed subsequently with saturated NaHCO₃ and saturated NaCl and driedover Na₂SO₄. The extract was evaporated and the residue was treated withtriethylamine (80 mL) in methanol (800 mL) at room temperature for 10hrs and evaporated. The residue was dissolved in chloroform anddeposited on a silica gel column. The product was eluted with a 10:1ethyl acetate:methanol mixture for the prodrugs containing C₉-C₁₆ alkylchains and 5:1 ethyl acetate:methanol mixture for the prodrugscontaining C₅-C₈ alkyl chains. Fractions containing the main productwere combined, evaporated and recrystallized from ethyl acetate,methanol, isopropanol or isopropanol-ether mixtures.

1.2 Results

Analytical data for exemplary compounds of the invention are providedbelow.

1.2.a N⁴-Amyloxycarbonyl-β-2′-deoxy-5,6-dihydro-5-azacytidine

The compound was obtained as a colorless solid usingn-amylchloroformate, m.p. 75-78°, (37% yield). ¹H NMR (300 MHz, DMSO-d₆)δ 0.858 (t, J=6.6 Hz, 3H, Me), 1.20-1.33 (m, 4H, (CH₂)₂), 1.50-1.58 (m,2H, OCH₂CH₂), 1.80-1.87 (m, 1H, H-2′), 1.95-2.04 (m, 1H, H-2″), 3.32 (brs, water), 3.42 (t, J=4.8 Hz, 2H, H-5′), 3.60-3.64 (m, 1H, H-4′), 3.94(t, J=6.8 Hz, 2H, OCH₂), 4.08-4.13 (m, 1H, H-3′), 4.55 (dd, J₁=18.4 Hz,J₂=10.4 Hz, 2H, 6-CH₂), 4.78 (t, J=5.2 Hz, 1H, OH-5′), 5.14 (d, J=4.4Hz, 1H, OH-3′), 6.01 (dd, J₁=8.0 Hz, J₂=6.4 Hz, 1H, H-1′), 9.4 (br s,1H, 5-NH), 10.1 (br s, 1H, NHCO). MS (ESI m/z) 345 (M+H)⁺.

1.2b N⁴-Hexyloxycarbonyl-β-2′-deoxy-5,6-dihydro-5-azacytidine

The compound was obtained as a colorless solid usingn-hexylchloroformate, m.p. 138-139°, (41% yield). ¹H NMR (300 MHz,DMSO-d₆) δ 0.858 (t, J=6.6 Hz, 3H, Me), 1.20-1.33 (m, 6H, (CH₂)₃),1.50-1.58 (m, 2H, OCH₂CH₂), 1.80-1.88 (m, 1H, 1-H-2′), 1.95-2.04 (m, 1H,H-2″), 3.32 (br s, water), 3.42 (t, J=4.4 Hz, 2H, H-5′), 3.60-3.64 (m,1H, H-4′), 3.94 (t, J=6.8 Hz, 2H, OCH₂), 4.08-4.13 (m, 1H, H-3′), 4.55(dd, J₁=18.4 Hz, J₂=10.4 Hz, 2H, 6-CH₂), 4.77 (br s, 1H, OH-5′), 5.14(d, J=4.0 Hz, 1H, OH-3′), 6.01 (dd, J₁=8.0 Hz, J₂=6.4 Hz, 1H, H-1′), 9.4(br s, 1H, 5-NH), 10.1 (br s, 1H, NHCO). MS (ESI m/z) 359 (M+H)⁺.

1.2.c N⁴-Heptyloxycarbonyl-β-2′-deoxy-5,6-dihydro-5-azacytidine

The compound was obtained as a colorless solid usingn-heptylchloroformate, m.p. 126-127.7°, (85% yield). ¹H NMR (300 MHz,DMSO-d₆) δ 0.857 (t, J=6.6 Hz, 3H, Me), 1.20-1.33 (m, 8H, (CH₂)₄),1.50-1.58 (m, 2H, OCH₂CH₂), 1.80-1.88 (m, 1H, H-2′), 1.96-2.04 (m, 1H,H-2″), 3.31 (br s, water), 3.42 (d, J=4.4 Hz, 2H, H-5′), 3.60-3.64 (m,1H, H-4′), 3.94 (t, J=6.8 Hz, 2H, OCH₂), 4.08-4.13 (m, 1H, H-3′), 4.56(dd, J₁=19.2 Hz, J₂=10.8 Hz, 2H, 6-CH₂), 4.77 (br s, 1H, OH-5′), 5.14(br s, 1H, OH-3′), 6.00 (dd, J₁=8.0 Hz, J₂=6.4 Hz, 1H, H-1′), 9.4 (br s,1H, 5-NH), 10.1 (br s, 1H, NHCO). MS (ESI m/z) 373 (M+H)⁺.

1.2.d N⁴-Octyloxycarbonyl-β-2′-deoxy-5,6-dihydro-5-azacytidine

The compound was obtained as a colorless solid usingn-octylchloroformate, m.p. 135-137°, (75% yield). ¹H NMR (300 MHz,DMSO-d₆) δ 0.856 (t, J=6.0 Hz, 3H, Me), 1.20-1.31 (m, 10H, (CH₂)₅),1.50-1.58 (m, 2H, OCH₂CH₂), 1.80-1.88 (m, 1H, H-2′), 1.95-2.04 (m, 1H,H-2″), 3.32 (br s, water), 3.43 (d, J=4.4 Hz, 2H, H-5′), 3.60-3.64 (m,1H, H-4′), 3.94 (t, J=6.8 Hz, 2H, OCH₂), 4.08-4.13 (m, 1H, H-3′), 4.55(dd, J₁=18.8 Hz, J₂=10.8 Hz, 2H, 6-CH₂), 4.78 (br s, 1H, OH-5′), 5.14(br s, 1H, OH-3′), 6.01 (dd, J₁=8.0 Hz, J₂=6.4 Hz, 1H, H-1′), 9.4 (br s,1H, 5-NH), 10.0 (br s, 1H, NHCO). MS (ESI m/z) 387 (M+H)⁺.

1.2.e N⁴— (2-Ethylhexyl)oxycarbonyl-β-2′-deoxy-5,6-dihydro-5-azacytidine

The compound was obtained as a colorless solid using(2-ethylhexyl)chloroformate, m.p. 118-120°, (55% yield). ¹H NMR (300MHz, DMSO-d₆) δ 0.82-0.89 (m, 6H, Me), 1.22-1.33 (m, 8H, CH₂), 1.46-1.54(m, 1H, CH), 1.80-1.87 (m, 111, H-2′), 1.95-2.04 (m, 1H, H-2″), 3.32 (brs, water), 3.42 (t, J=5.0 Hz, 2H, H-5′), 3.60-3.64 (m, 1H, H-4′),3.84-3.90 (m, 2H, OCH₂), 4.08-4.13 (m, 1H, H-3′), 4.56 (dd, J₁=19.0 Hz,J₂=10.6 Hz, 2H, 6-CH₂), 4.77 (t, J=5.4 Hz, 1H, OH-5′), 5.14 (d, J=4.0Hz, 1H, OH-3′), 6.01 (dd, J₁=8.0 Hz, J₂=6.0 Hz, 1H, H-1′), 9.4 (br s,1H, 5-NH), 10.1 (br s, 1H, NHCO). MS (ESI m/z) 387 (M+H)⁺.

1.2.f N⁴-Nonyloxycarbonyl-β-2′-deoxy-5,6-dihydro-5-azacytidine

The compound was obtained as a colorless solid usingn-nonylchloroformate, m.p. 137-138.4°, (75% yield). ¹H NMR (300 MHz,DMSO-d₆) δ 0.855 (t, J=6.0 Hz, 3H, Me), 1.20-1.31 (m, 12H, (CH₂)₆),1.50-1.58 (m, 2H, OCH₂CH₂), 1.80-1.88 (m, 1H, H-2′), 1.95-2.04 (m, 1H,H-2″), 3.32 (br s, water), 3.42 (d, J=4.4 Hz, 211, H-5′), 3.60-3.64 (m,1H, H-4′), 3.94 (t, J=6.8 Hz, 2H, OCH₂), 4.08-4.13 (m, 1H, H-3′), 4.55(dd, J₁=10.8 Hz, J₂=18.8 Hz, 2H, 6-CH₂), 4.78 (br s, 1H, OH-5′), 5.14(br s, 1H, OH-3′), 6.00 (dd, J₁=8.0 Hz, J₂=6.4 Hz, 1H, H-1′), 9.4 (br s,1H, 5-NH), 10.0 (br s, 11-1, NHCO). MS (ESI m/z) 401 (M+H)⁺.

1.2.g N⁴-Decyloxycarbonyl-β-2′-deoxy-5,6-dihydro-5-azacytidine

The compound was obtained as a colorless solid usingn-decylchloroformate, m.p. 124-126°, (86% yield). ¹H NMR (300 MHz,DMSO-d₆) δ 0.853 (t, J=6.0 Hz, 3H, Me), 1.20-1.31 (m, 14H, (CH₂)₇),1.50-1.58 (m, 2H, OCH₂CH₂), 1.80-1.87 (m, 1H, H-2′), 1.95-2.04 (m, 1H,H-2″), 3.32 (br s, water), 3.42 (t, J=4.8 Hz, 2H, H-5′), 3.60-3.64 (m,1H, H-4′), 3.94 (t, J=6.8 Hz, 2H, OCH₂), 4.08-4.13 (m, 1H, H-3′), 4.55(dd, J₁=18.4 Hz, J₂=10.4 Hz, 2H, 6-CH₂), 4.78 (t, J=4.8 Hz, 1H, OH-5′),5.14 (d, J=4.4 Hz, 1H, OH-3′), 6.01 (dd, J₁=8.0 Hz, J₂=6.4 Hz, 1H,H-1′), 9.4 (br s, 1H, 5-NH), 10.0 (br s, 1H, NHCO). MS (ESI m/z) 415(M+H)⁺.

1.2.h N⁴-Dodecyloxycarbonyl-β-2′-deoxy-5,6-dihydro-5-azacytidine

The compound was obtained as a colorless solid usingn-dodecylchloroformate, m.p. 138-140°, (76% yield). ¹H NMR (300 MHz,DMSO-d₆) δ 0.853 (t, J=7.0 Hz, 3H, Me), 1.20-1.31 (m, 18H, (CH₂)₉),1.50-1.60 (m, 2H, OCH₂CH₂), 1.80-1.87 (m, 1H, H-2′), 1.95-2.04 (m, 1H,H-2″), 3.31 (br s, water), 3.42 (t, J=5.2 Hz, 2H, H-5′), 3.60-3.64 (m,1H, H-4′), 3.94 (t, J=6.8 Hz, 2H, OCH₂), 4.08-4.13 (m, 1H, H-3′), 4.55(dd, J₁=18.4 Hz, J₂=10.4 Hz, 2H, 6-CH₂), 4.78 (t, J=4.8 Hz, 1H, OH-5′),5.14 (d, J=4.4 Hz, 1H, OH-3′), 6.01 (dd, J₁=8.0 Hz, J₂=6.4 Hz, 1H,H-1′), 9.4 (br s, 1H, 5-NH), 10.0 (br s, 1H, NHCO). MS (ESI m/z) 443(M+H)⁺.

1.2.i N⁴-Hexadecyloxycarbonyl-β-2′-deoxy-5,6-dihydro-5-azacytidine

The compound was obtained as a colorless solid usingn-hexadecylchloroformate, m.p. 119-123°, (73% yield). ¹H NMR (300 MHz,DMSO-d₆) δ 0.853 (t, J=6.0 Hz, 3H, Me), 1.20-1.31 (m, 26H, (CH₂)₁₃),1.50-1.58 (m, 2H, OCH₂CH₂), 1.80-1.88 (m, 1H, H-2′), 1.95-2.04 (m, 1H,H-2″), 3.31 (br s, water), 3.42 (t, J=4.8 Hz, 2H, H-5′), 3.59-3.64 (m,1H, H-4′), 3.94 (t, J=6.4 Hz, 2H, OCH₂), 4.08-4.13 (m, 1H, H-3′), 4.56(dd, J₁=18.8 Hz, J₂=10.8 Hz, 2H, 6-CH₂), 4.78 (t, J=5.2 Hz, 1H, OH-5′),5.14 (d, J=4.0 Hz, 1H, OH-3′), 6.01 (dd, J₁=7.6 Hz, J₂=6.8 Hz, 1H,H-1′), 9.4 (br s, 1H, 5-NH), 10.0 (br s, 1H, NHCO). MS (ESI m/z) 499(M+H)⁺.

1.2.j N⁴-Heptyloxycarbonyl-α-2′-deoxy-5,6-dihydro-5-azacytidine

The compound was obtained as a colorless solid usingα-2′-deoxy-5,6-dihydro-5-azacytidine and n-heptylchloroformate, m.p.77-78°, (30% yield). ¹H NMR (300 MHz, DMSO-d₆) δ 0.857 (t, J=6.8 Hz, 3H,Me), 1.20-1.33 (m, 8H, (CH₂)₄), 1.50-1.58 (m, 2H, OCH₂CH₂), 1.68-1.76(m, 1H, H-2′), 2.38-2.47 (m, 1H, H-2″), 3.31 (br s, water), 3.33-3.40(m, 2H, H-5′), 3.82-3.87 (m, 1H, H-4′), 3.94 (t, J=6.6 Hz, 2H, OCH₂),4.11-4.16 (m, 1H, H-3′), 4.60 (d, J=11.2 Hz, 1H, 6-CH_(a)), 4.75 (t,J=5.6 Hz, 1H, OH-5′), 4.77 (d, J=11.2 Hz, 1H, 6-CH_(b)), 5.24 (d, J=3.6Hz, 1H, OH-3′), 5.97 (dd, J₁=8.0 Hz, J₂=4.8 Hz, 1H, H-1′), 9.4 (br s,1H, 5-NH), 9.9 (br s, 1H, NHCO). MS (ESI m/z) 373 (M+H)⁺.

Example 2 Experimental Conditions for the Bioavailability Studies of theCompounds in Rats

This study tested the bioavailability of different test compounds inrats, by oral gavage administration, at prescribed dose levels.

The animals tested in this study were Rattus norvegicus, CD strain, fromCharles River Canada (Montreal, Canada). Six males were included in eachtest group. Their weight ranged from 200 to 250 grams before fasting.The weight variation in animals at the initiation of the study did notexceed plus or minus 20% of the mean weight.

Rats were individually housed in Nalgene rat cages with stainless steelcovers. The animal room environment was controlled and monitored daily.The temperature ranged from 18 to 26° C. and the relative humidityranged from 30%-70%. The photocycle was 12 hours light and 12 hoursdark. All animals were submitted to a general physical examination andonly those found healthy were admitted for the study. All selectedanimals were given two days to adjust to laboratory conditions beforethe study began. Teklad rodent Diet and water were constantly availableto the rats throughout the acclimatization and study periods.

Forty-eight hours prior to the initiation of the study, animals wererandomly selected and two subsets of 3 male rats per group were formed.Due to the large volume of blood taken, collections will be done byalternating timepoints between subsets.

All animals were fasted for twelve hours prior to the initiation of thestudy. At the study initiation, respective groups of rats received thecompound of the invention by oral gavage at a dose of 4.0 mL/kg.

Blood was collected from the orbital sinus of animals from each group atthirty minutes and at 1, 2, 4, and 12 hours after dosing. At each bloodcollection time point, three rats per group were bled.

Example 3 Pharmacokinetic Data for the Compounds of the Invention inRats

DHAdC (5,6-dihydro-5-aza-2′-deoxycytidine) and the prodrugsC₇-DHAdC(N⁴-heptyloxycarbonyl-DHAdC) andC₉-DHAdC(N⁴-nonyloxycarbonyl-DHAdC) were administered to rats under theconditions described in Example 2. Oral and IV dosages of DHAdC, as wellas oral dosages of prodrugs C₇-DHAdC and C₉-DHAdC were recorded. Therats were administered 100 mg/kg DHAdC or DHAdC equivalent in the caseof prodrugs. The maximum concentration (C_(max)), clearance (AUC∞),half-life (T_(1/2)), and % Oral Bioavailability were recorded and arepresented in Table 1:

TABLE 1 DHAdC DHAdC C₇-DHAdC C₉-DHAdC (IV) (oral) (oral) (oral) C_(max)(ng/mL) 40,034 1,858 5,577 5,550 AUC∞ (ng- 74,975 8,538 32,101 26,472hr/mL) T_(1/2) (hr) 1.8 0.8 3.9 3.6 Oral N/A 11 43 35 bioavailability(%)

Example 4 Experimental Conditions for the Bioavailability Studies of theCompounds in Dogs

This study tested the bioavailability of different test compounds indogs, by intravenous or oral gavage administration, at prescribed doselevels.

The animals tested in this study were Canis familiaris, from HarlanSprague Dawley (Indianapolis, Ind.). Four dogs (2 males and 2 females),with an average age of seven months, were included in each test group.The dogs were identified by ear tattoos, and given a fourteen dayacclimatization period prior to the commencement of testing.

Dogs were group housed but separated by sex. The animal room environmentwas controlled and monitored daily. The temperature ranged from 18 to29° C. and the relative humidity ranged from 30%-70%. The photocycle was12 hours light and 12 hours dark. All animals were submitted to ageneral physical examination and only those found healthy were admittedfor the study. All selected animals were given two days to adjust tolaboratory conditions before the study began. Teklad Dog Diet was fedtwice daily and water was constantly available to the dogs throughoutthe acclimatization and study periods.

Twenty-four hours prior to the initiation of the study, animals wererandomly selected into two groups with 1 male and 1 female in eachgroup. At the study initiation, the respective groups of dogs thenreceived a compound of the invention by intravenous injection or oralgavage administration at 0.5 mL/kg.

Blood was collected from the v. cephalica antebrachii of each of the twoanimals prior to study initiation, at thirty minutes and at 1, 2, 4, and12 hours after dosing.

Example 5 Pharmacokinetic Data for the Compounds of the Invention inDogs

DHAdC (5,6-dihydro-5-aza-2′-deoxycytidine) and the prodrugsC₇-DHAdC(N⁴-heptyloxycarbonyl-DHAdC),C₈-DHAdC(N⁴-octyloxycarbonyl-DHAdC), andC₉-DHAdC(N⁴-nonyloxycarbonyl-DHAdC) were administered to dogs under theconditions described in Example 4. IV dosages of DHAdC, as well as oraldosages of prodrugs C₇-DHAdC, C₈-DHAdC, and C₉-DHAdC were recorded. Thedogs were administered 50 mg/kg DHAdC or DHAdC equivalent in the case ofprodrugs. The maximum concentration (C_(max)), clearance (AUC∞),half-life (T_(1/2)), and % Oral Bioavailability were recorded and arepresented in Table 2:

TABLE 2 DHAdC C₇-DHAdC C₈-DHAdC C₉-DHAdC (IV) (oral) (oral) (oral)C_(max) (ng/mL) 76,333 11,282 5,172 8,932 AUC∞ (ng- 156,058 59,37717,845 54,540 hr/mL) T_(1/2) (hr) 2.1 2.1 2.8 2.2 Oral N/A 51 11 36bioavailability (%)

Example 6 Experimental Conditions for the Lipophilicity Study

This study tested the lipophilicity of different test compounds.

The compounds were first dissolved in a 60% methanol/40% water (v/v)solution containing 1% DMSO and brought to a concentration of 100 μM.The optimal detection wavelength was determined and calibration studieswere performed with the solution. After calibration, the compounds wereplaced into n-octanol-PBS buffer at pH 7.4 at RT. The mixture was shakenand allowed to equilibrate for 60 minutes. The amount of compound in thePBS buffer phase was then determined by HPLC/UV-Vis. The amount ofcompound in n-octanol was determined by subtracting the original amountfrom the amount in the PBS buffer phase. This experiment was conductedfor three samples. These three samples were then each corrected forvolume and the results were averaged. Log D was calculated as the Log₁₀of the amount of compound in the n-octanol phase divided by the amountof compound in the buffer phase.

Example 7 Lipophilicity Data for the Compounds of the Invention

Lipophilicity is a major structural factor that influences thepharmacokinetic and pharmacodynamic behavior of compounds. In thisassay, prodrugs C₇-DHAdC, C₈-DHAdC, and C₉-DHAdC were partitionedbetween n-octanol and phosphate buffered saline (PBS). The log D valuesof the prodrugs are recorded in Table 3:

TABLE 3 log D n-Octanol Compound I.D. PBS pH 7.4 Test Concentration (uM)100 um C₇-DHAdC 2.04 C₈-DHAdC 2.56 C₉-DHAdC 3.16

A value between 2.0 and 2.5 might be considered optimal.

1. A compound having a structure according to Formula I:

wherein a is 1; R² is (═O), such that b is 0; R⁴ is NR⁷R⁸; R⁶ is H; R⁷,R⁸, and R⁵ are members independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl and astructure according to formula IV or formula V:

wherein each of R¹⁴ and R¹⁵ is independently selected from substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted acyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl, anamino acid, and a peptide comprising between 2 and 5 amino acids, andwherein if R⁸ is a structure according to Formula IV or Formula V, thenR⁷ is H; and R¹ is a structure according to Formula II:

wherein R⁹ is H; and R¹⁰ and R¹¹ are —OH wherein at least one memberselected from R⁵, R⁷, and R⁸, together with the nitrogen atom to whichit is covalently bonded, is selected from a carbamate and a urea.
 2. Thecompound according to claim 1, wherein R⁵, R⁷, and R⁸ are independentlyselected from H and a structure according to Formula IV or Formula V. 3.The compound according to claim 2, wherein each of R¹⁴ and R¹⁵ areindependently substituted or unsubstituted alkyl.
 4. The compoundaccording to claim 3, wherein each of R¹⁴ and R¹⁵ are independentlyunsubstituted alkyl.
 5. The compound according to claim 3, wherein eachof R¹⁴ and R¹⁵ are independently unsubstituted C₅-C₁₆ alkyl.
 6. Thecompound according to claim 5, wherein each of R¹⁴ and R¹⁵ areindependently selected from n-pentyl, n-hexyl, n-heptyl, n-octyl,2-ethyl-hexyl, n-nonyl, n-decyl, n-dodecyl and n-hexadecyl.
 7. Thecompound according to claim 5, wherein R¹⁴ or R¹⁵ is n-heptyl.
 8. Thecompound according to claim 2, wherein R⁸ is a structure according toFormula IV or Formula V; and R⁵ is H.
 9. The compound according to claim8, wherein each of R¹⁴ and R¹⁵ are independently selected from n-pentyl,n-hexyl, n-heptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n-decyl, n-dodecyland n-hexadecyl.